DESCRIPTION The project is focused on functional and evolutionary analysis of proteins involved in iron-sulfur (FeS) cluster assembly in amitochondriate parasites of importance in human (Trichonionas vaginalis, Entamoeba histolytica, Girdia lamblia) and veterinary (Tritrichomonas foetus) medicine. FeS clusters are present in numerous proteins that have essential catalytic, redox and regulatory properties. In "typical" eukaryotic cells, FeS proteins are present mainly in mitochondria, while in amitochondriate anaerobic eukaryotes, proteins possessing FeS clusters have been found in cytosol (Giardia, Entamoeba) or in hydrogenosomes (trichomonads). Growing molecular data, including analysis of FeS proteins indicate, that "amitochondriate" phenotype should not be considered as a primitive, and that the ancestors of amitochondriate protists experienced the endosymbiotic event that led to the establishment of mitochondrion. If so, formation of FeS clusters required for function both mitochondrial and "amitochondrial" proteins, should be mediated by a common process. This hypothesis will be tested by: (I) Identification of components involved in FeS cluster assembly based on sequence homology with those present in prokaryotic organisms and mitochondria of eukaryotes, (2) comparative sequencing of selected proteins, (3) their biochemical and functional characterization, and (4) establishment of their subcellular localization. Initial experiments will focus on sequencing, expression and functional analysis of "amitochondriate" NifS homologs that catalyze the formation of elemental sulfur required for FeS cluster assembly, and NifU that provides iron. Analysis of other components of FeS assembly machinery (NifA, ferredoxin, NAD(P)H:feredoxin oxidoreductase) will be included as the project proceeds. The proposed study of FeS clusters assembly will contribute to understanding of the physiology of amitochondriate parasites thus it can provide new chemotherapeutic approaches for dealing with parasitic infections. Moreover, the study of FeS in amitochondrial cells, particularly in hydrogenosomes, could reveal unique information relevant to mitochondrial iron metabolism in humans that recently attracted attention of biomedical research.